Telomeres are terminal structural elements found at the end of chromosomes [Muller, The Collecting Net-Woods Hole, 13:181-195 (1939)] that protect natural double-stranded DNA ends from degradation, fusion, and recombination with chromosome-internal DNA [McClintock, Genetics, 26:234-282 (1941); Lundblad et al., Cell, 87:369-375 (1996)]. Telomeres are also thought to play a role in the architecture of the nucleus [Agard et al., Nature, 302:676-681 (1983); Rabl, Morphol. J, 10:214-330 (1885)], and to provide a solution to the end-replication problem that arises as a consequence of successive replication of linear DNA by DNA polymerases which would otherwise result with progressively shorter terminal sequences [Watson, Nature, 239:197-201 (1972)]. In tetrahymena, impaired telomere function leads to a defect in cytokinesis and to cell death [Yu et al., Nature, 344:126-132 (1990)]. Similarly, in yeast, loss of a single telomere results in cell cycle arrest and chromosome instability [Sandell and Zakian, Cell, 75:729-741 (1993)] and cells undergoing generalized telomere shortening eventually senesce [Lundblad and Szostak, Cell, 57:633-643 (1989); Singer and Gottschling, Science, 266:404-409 (1994)].
A ribonucleoprotein reverse transcriptase, telomerase, can elongate telomeres using an internal RNA component as template for the addition of the appropriate G-rich sequence to the 3' telomere termini [Greider and Blackburn, Cell, 43:405-413 (1985)]. This activity is thought to compensate for the inability of polymerases to replicate chromosome ends, but other mechanisms of telomere maintenance may operate as well [Pluta et al., Nature, 337:429-433 (1989)].
Telomeres contain a tandem array of repeat sequences, typically five to eight base pairs long, that are G-rich in the strand that extends to the end of the chromosome DNA. These repeat units appear to be both necessary and sufficient for telomere function [Lundblad et al., 1989, supra; Szostak et al., Cell, 36:459-568 (1982)]. All telomeres of a single genome are composed of the same repeats and these sequences are highly conserved across species. For instance, Oxytricha chromosomes terminate in TTTMGGGG repeats [Klobutcher et al., Proc. Natl. Acad. Sci. USA, 78:3015-3019 (1981)], Tetrahymena utilizes an array of (TTGGGG).sub.n. [Blackburn et al., J. Mol. Biol., 120:33-53 (1978)], and plant chromosomes carry the sequence (TTTAGGG).sub.n [Richards et al., Cell, 53:127-136 (1988)].
Telomeres of trypanosomes and all vertebrates, including mammals, contain the repeat sequence TTAGGG [Blackburn et al., Cell, 36:447-458 (1984); Brown, Nature, 338:774-776 (1986); Cross et al., Nature, 338:771-774 (1989); Moyzis et al., Proc. Natl. Acad. Sci. USA, 85:6622-6626 (1988); Van der Ploeg et al., Cell, 36:459-468 (1984)]. This 6 bp sequence is repeated in long tandem arrays at the chromosome ends, which may be as long as 100 kb in the mouse, and varies from 2 to 30 kb in humans [Zhong et al., Mol. Cell. Biol., 13:4834-4943 (1992)].
During the development of human somatic tissue, telomeres undergo progressive shortening; in contrast, sperm telomeres increase with donor age [Broccoli et al., Proc. Natl. Acad. Sci. USA, 92:9082-9086 (1995); de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994)]. Most if not all human somatic tissue chromosomes lose terminal TTAGGG repeats with each division, e.g., about 15-40 bp per year in the skin and blood. It is unclear what effect this diminution has since human telomeres are between 6-10 kb at birth. On the other hand, it is not yet known how many kilobases of TIAGGG repeats are necessary for optimal telomere function.
Primary human fibroblasts grown in culture lose about 50 bp of telomeric DNA per doubling (PD) before they stop dividing at a senescence stage [Allsopp et al., Proc. Natl. Acad. Sci. USA, 89:10114-10118 (1992)]. Importantly, there is an excellent correlation between the number of divisions that the cells go through and their initial telomere length. Indeed, it has been suggested that the correlation represents a molecular clock, which limits the potential of primary cells to replicate [Harley et al., Nature (London), 345:458-460 (1990); Harley et al., Exp. Gerontol, 27:375-382 (1992)]. Thus, immortalization of human somatic cells appears to involve a mechanism to halt telomere shortening. Recently Bodner et al. [Science, 279:349-352 (1998)] have reported a direct correlation between the introduction of telemorase into a cell and the proliferative life-span of the cell.
Changes in telomeric dynamics also appear to play a role in the malignant transformation of human cells [Broccoli et al., 1995, supra]. For example, telomeres of tumor cells are generally significantly shorter than those of the corresponding normal cells. Telomerase activation appears to be an obligatory step in the immortalization of human cells and in particular, in ovarian carcinoma [de Lange, 1994, supra]. Hanish et al. [Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)] examined the requirements for the formation of human telomeres from TTAGGG seeds, and found that telomere formation was not correlated with the ability of human telomerase to elongate telomeric sequences in vitro, and did not appear to be a result of homologous recombination. Rather, the sequence dependence of telomere formation matched the in vitro binding requirements for TRF, a telomeric TTAGGG repeat binding protein that is associated with human and mouse telomeres in interphase and in mitosis.
Indeed, the only known protein components of mammalian telomeres are the TRF proteins, duplex TTAGGG repeat binding factors that are localized at telomeres in interphase and metaphase chromosomes [Zhong et al., 1992, supra; Chong et al., Science, 270:1663-1667 (1995); Luderus et al., J. Cell Biol., 135:867-881 (1996); Broccoli et al., Hum. Mol. Genetics, 6:69-76 (1997); see Smith and de Lange, Trends in Genetics, 13:21-26 (1997) for review]. Human TRF1 (hTRF1) is a low-abundance activity found in nuclear extracts from all human cells and tissues and a similar activity is present in other vertebrates [Zhong et al., 1992, supra; Chong et al., 1995, supra (U.S. patent application Ser. No. 08/519,103 filed Aug. 25, 1995, hereby incorporated by reference in its entirety)]. TRF2 (also referred to as orf2) has been identified as a TRF1 homolog. [Bilaud et al., Nucl. Acids Res., 24:1294-1303 (1996) (U.S. patent application Ser. No. 08/938,052 filed Sep. 26, 1997, hereby incorporated by reference in its entirety)]. Similar duplex telomeric DNA binding proteins in yeasts have been implicated in telomere length control, telomere stability, and telomeric silencing [reviewed in Shore, Trends Gen., 10:408-412 (1994); Zakian, Saccharomyces telomere. function, structure and replication, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 107-138 (1995a); see also McEachern and Blackburn, Nature, 376:403-409 (1995); Krauskopf and Blackburn, Nature, 383:354-357 (1996)].
TRF1 has DNA binding properties in vitro that are consistent with its presence along the double-stranded telomeric repeat array at chromosome ends. TRF1 binds efficiently to arrays of duplex TTAGGG repeats, irrespective of the presence of a DNA terminus [Zhong et al., 1992, supra]. Single-stranded telomeric DNA is not an effective TRF1 substrate and neither are heterologous telomeric sequences, such as double-stranded arrays of TTGGGG, TTAGGC, TTTAGGG, TTAGGGGG, and TAGGG repeats [Zhong et al., 1992, supra; Hanish et al., 1994, supra; Chong et al., 1995, supra]. This sequence specificity of TRFs match the sequence requirements for de novo telomere formation in human cells, suggesting that TRF proteins are involved in this process [Hanish et al., 1994, supra].
Interestingly, TRF1 binding is stimulated by longer repeat arrays with 6 or 12 repeat providing a better binding substrate than 3 repeats [Zhong et al., 1992, supra]. Since DNA fragments with 3, 6, or 12 telomeric repeats each bind exactly the same protein mass, this enhancement is not due to cooperative interactions between multiple TRF1 binding units. The minimal TRF1 binding site and the mechanism by which this protein differentiates between telomeric arrays of different length remain to be deter-mined.
Mouse and human TRF1 are novel proteins with three recognizable domains: an acidic domain at the N-terminus, a conserved TRF-specific domain, and a C-terminal domain with strong homology to the DNA binding domain of Myb oncoproteins (see FIG. 1; [Chong et al., 1995, supra; U.S. patent application Ser. No. 08/519,103 filed Aug. 25, 1995]). c-Myb oncoproteins are transcriptional activators that carry three imperfect 50 amino acid repeats, two of which are required for DNA binding. In c-Myb, the two Myb repeats fold into helix-turn-helix motifs that are closely packed on the DNA such that their recognition helices together contact a single short PyAACNG site [Ogata et al., Cell, 79:629-648 (1994)]. In other Myb-related DNA binding proteins, Myb repeats have been found in four configurations: three tandem repeats (for instance, in the yeast protein BAS1 [Hovring et al., J. Biol. Chem., 296:17663-17669 (1994)]), two tandem repeats (in many plant transcription factors [Ramachandran et al., Curr. Op. Genet. Dev., 4:642-646 (1994)]), and in the fission yeast protein cdc5 [Ohi et al., EMBO J., 13:471-483 (1994)], two repeats separated by a linker (in the yeast proteins Reb1p and Rap1p (Repressor/Activator protein 1) and in the mouse protein MIDA1 [Morrow et al., Mol. Cell. Biol., 13:1173-1182 (1993); Konig et al., Cell, 85:125-136 (1996); Sitzmann et al., Oncogene, 12:1889-1894 (19 96)]; and single Myb repeats (in several yeast, plant, Drosophila, and mouse proteins [England et al., Proc. Natl. Acad. Sci. USA, 89:683-687 (1991); Brigati et al., Mol. Cell. Biol., 13:1306-1314 (1993); da Costa e Silva et al., The Plant Journal, 4:125-135 (1993); Baranowskij et al., EMBO J., 13:5383-5392 (1994); Lugert and Werr, Plant Molecular Biology, 25:493-506 (1994); Stokes and Perry, Mol. Cell. Biol., 15:2745-2753 (1995)].
The group of proteins with one Myb repeat, which includes TRF1 and TRF2, had presented a conundrum, since in other Myb-related factors at least two Myb repeats are required for DNA binding [Henry et al., Proc. Natl. Acad. Sci. USA, 18:2617-2623 (1990); Saikumar et al., Proc. Natl. Acad. Sci. USA, 87:8452-8456 (1990); Hovring et al., 1994, supra].
Remarkably, TRF1 evolved rapidly [Broccoli et al., 1997, supra] and does not show significant amino acid identity with Rap1p, the major duplex telomeric DNA binding protein of the yeasts Saccharomyces cerevisiae [Shore, 1994, supra] and Kluyveromyces lactis [Larson et al., Gene, 150:35-41 (1994); Krauskopf and Blackburn, 1996, supra]. Yet, the yeast and mammalian telomeric proteins appear to be distantly-related, since both carry Myb-related DNA binding domains [Konig et al., 1996, supra]. Rap1p contains two Myb repeats, which, separated by a 40 amino acid linker, dock onto two GGTGT sequences that are separated by 3 bp. Since Rap1p and c-Myb bind DNA differently (Ogata et al., 1994, supra; Konig et al., 1996, supra), no a priori predictions can be made on the DNA binding mode of TRF1 and TRF2. Indeed the fact that TRF1 and TRF2 contain only a single Myb motif [Chong et al., 1995, supra] points to a crucial difference in the way these factors bind to DNA compared with c-Myb and Rap1p.
Mammalian telomeres show a species-specific length setting suggesting a regulatory mechanism that controls telomere length in the germline. Telomere length control is also evident from the stability of telomeres in telomerase expressing cells lines and from the observation that newly-formed telomeres acquire a length appropriate for the host cell. The latter observation suggests that cells monitor and modulate the length of individual telomeres, a process likely to involve a protein such as a TRF, that binds to the duplex telomeric repeat region at mammalian chromosome ends.
Therefore, there is a need to identify agents that can modify and/or control telomere lengthening and/or telomere maintenance. More particularly, there is a need to identify an agent that can modify or/inhibit the activity of TRFs either individually, such as an inhibitor to TRF1 or TRF2 or a more general agent that inhibits two or more TRFs. Furthermore, there is a need to characterize such an agent.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.